Dear all, I have tried the procedure recommended by Zbyszek, expanding data from a higher symmetry and keeping the R-free set. But the map for third molecule (new molecule placed) are still very bad, even when a tried to reprocess data in P1 or P2 (P 1 21 1). The previous placed molecule (present in P2 21 21 ASU) and its symmetry related on P21 shows a very good map, but the third molecule are almost completely wrong (~50 residues in 470 are placed in quite good map) and map does not have connectivity to build a new molecule (even in lower sigmas, 0.8-1.0). I have tried automatic model building (AutoBuild and ARP/wARP) but they cannot build anything that make some sense or build a random chains without any sense.
I do not have an extensive knowledge of crystallography, but I have been thinking about some questions: If the third molecule (the bad one) is lying on the 2-fold symmetry axis on P 2 21 21, and since it does not have an intrinsic 2-fold symmetry axis (like protein molecule), how can I merge the structure factors (or intensities) related by symmetry and expand to lower symmetry afterwards? In this case the molecule lying on the 2-fold symmetry axis will have the structure factors wrongly merged, since the molecule is not symmetric, is it ok? If the third molecule is lying on the 2-fold symmetry axis on P 2 21 21, and only another two molecules can be related by the crystallographic symmetry, is it a case of pseudo-symmetry? But in this case, the third molecule is disordered in the crystal packing (as Zbyszek said), and probably have a long range disorder, because I cannot get a good maps for this third molecule even in P1. (pseudo-symmetry + order/disorder????). And a more philosophical question… what is the problem in process data in a lower symmetry? Are there mathematical/statistical problems related that can lead to “false-good” data? I put a new .pdf file (ccp4bb_maps_P21.pdf) with map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps_P21.pdf I am sorry for so many questions and thanks in advance. Cheers, Andrey 2013/3/20 Jrh <[email protected]> > Dear Zbyszek, > I am concerned that the unmerged data would be bypassed and not preserved > in your recommendation. I also find it counter intuitive that the merged > data would then be unmerged into a lower symmetry and be better than the > unmerged data; there is I imagine some useful reference or two you can > direct me to that may well correct my lack of understanding. Thirdly I > think this a very likely useful case to preserve the raw diffraction images. > All best wishes, > John > > Prof John R Helliwell DSc > > > > On 19 Mar 2013, at 14:37, Zbyszek Otwinowski <[email protected]> > wrote: > > > It is a clear-cut case of crystal packing disorder. The tell-tale sign is > > that data can be merged in the higher-symmetry lattice, while the number > > of molecules in the asymmetric unit (3 in P21) is not divisible by the > > higher symmetry factor (2, by going from P21 to P21212). > > From my experience, this is more likely a case of order-disorder than > > merohedral twinning. The difference between these two is that structure > > factors are added for the alternative conformations in the case of > > order-disorder, while intensities (structure factors squared) are added > in > > the case of merohedral twinning. > > > > Now an important comment on how to proceed in the cases where data can be > > merged in a higher symmetry, but the structure needs to be solved in a > > lower symmetry due to a disorder. > > > > !Such data needs to be merged in the higher symmetry,assigned R-free > flag, > > and THEN expanded to the lower symmetry. Reprocessing the data in a lower > > symmetry is an absolutely wrong procedure and it will artificially reduce > > R-free, as the new R-free flags will not follow data symmetry! > > > > Moreover, while this one is likely to be a case of order-disorder, and > > these are infrequent, reprocessing the data in a lower symmetry seems to > > be frequently abused, essentially in order to reduce R-free. Generally, > > when data CAN be merged in a higher symmetry, the only proper procedure > in > > going to a lower-symmetry structure is by expanding these higher-symmetry > > data to a lower symmetry, and not by rescaling and merging the data in a > > lower symmetry. > > > > Zbyszek Otwinowski > > > >> Dear all, > >> We have solved the problem. Data processing in P1 looks better (six > >> molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules > >> in > >> ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round > >> of refinement (without put waters, ligands, etc.). > >> > >> Indeed, there were one more molecule in ASU, but the over-merged data in > >> an orthorhombic lattice hid the correct solution. > >> > >> Thank you very much for all your suggestions, they were very important > to > >> solve this problem. > >> > >> Cheers, > >> > >> Andrey > >> > >> 2013/3/15 Andrey Nascimento <[email protected]> > >> > >>> *Dear all,* > >>> > >>> *I have collected a good quality dataset of a protein with 64% of > >>> solvent > >>> in P 2 21 21 space group at 1.7A resolution with good statistical > >>> parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; > >>> Complet.=93% > >>> Redun.=2.4, the overall values are better than last shell). The > >>> structure > >>> solution with molecular replacement goes well, the map quality at the > >>> protein chain is very good, but in the final of refinement, after > >>> addition > >>> of a lot of waters and other solvent molecules, TLS refinement, etc. > ... > >>> the Rfree is a quite high yet, considering this resolution > >>> (1.77A).(Rfree= > >>> 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a > lower > >>> symmetry space group (P21), but I got the same problem, and I tried all > >>> possible space groups for P222, but with other screw axis I can not > even > >>> solve the structure.* > >>> > >>> *A strange thing in the structure are the large solvent channels with a > >>> lot of electron density positive peaks!? I usually did not see too many > >>> peaks in the solvent channel like this. This peaks are the only reason > >>> for > >>> these high R's in refinement that I can find. But, why are there too > >>> many > >>> peaks in the solvent channel???* > >>> > >>> *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map > >>> figures in this link: > https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* > >>> > >>> * > >>> * > >>> > >>> *Do someone have an explanation or solution for this?* > >>> > >>> * * > >>> > >>> *Cheers,* > >>> > >>> *Andrey* > >>> > >> > > > > > > Zbyszek Otwinowski > > UT Southwestern Medical Center at Dallas > > 5323 Harry Hines Blvd. > > Dallas, TX 75390-8816 > > Tel. 214-645-6385 > > Fax. 214-645-6353 >
